Nonribosomal Peptides, Key Biocontrol Components for Pseudomonasfluorescens In5, Isolated from a Greenlandic Suppressive Soil

Charlotte F. Michelsen,a* Jeramie Watrous,b,c,d Mikkel A. Glaring,a Roland Kersten,b,c,d Nobuhiro Koyama,e Pieter C. Dorrestein,b,c,d

Peter Stougaarda

Department of Plant and Environmental Sciences, University of Copenhagen, Copenhagen, Denmarka; Department of Pharmacology, University of California at SanDiego, La Jolla, California, USAb; Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, California, USAc; Skaggs School of Pharmacyand Pharmaceutical Sciences, University of California at San Diego, La Jolla, California, USAd; Graduate School of Pharmaceutical Sciences, Kitasato University, Tokyo,Japane

* Present address: Charlotte F. Michelsen, Department of Systems Biology, Technical University of Denmark, Kongens Lyngby, Denmark.

ABSTRACT Potatoes are cultivated in southwest Greenland without the use of pesticides and with limited crop rotation. Despitethe fact that plant-pathogenic fungi are present, no severe-disease outbreaks have yet been observed. In this report, we documentthat a potato soil at Inneruulalik in southern Greenland is suppressive against Rhizoctonia solani Ag3 and uncover the suppres-sive antifungal mechanism of a highly potent biocontrol bacterium, Pseudomonas fluorescens In5, isolated from the suppressivepotato soil. A combination of molecular genetics, genomics, and matrix-assisted laser desorption ionization–time of flight(MALDI-TOF) imaging mass spectrometry (IMS) revealed an antifungal genomic island in P. fluorescens In5 encoding two non-ribosomal peptides, nunamycin and nunapeptin, which are key components for the biocontrol activity by strain In5 in vitro andin soil microcosm experiments. Furthermore, complex microbial behaviors were highlighted. Whereas nunamycin was demon-strated to inhibit the mycelial growth of R. solani Ag3, but not that of Pythium aphanidermatum, nunapeptin instead inhibitedP. aphanidermatum but not R. solani Ag3. Moreover, the synthesis of nunamycin by P. fluorescens In5 was inhibited in the pres-ence of P. aphanidermatum. Further characterization of the two peptides revealed nunamycin to be a monochlorinated9-amino-acid cyclic lipopeptide with similarity to members of the syringomycin group, whereas nunapeptin was a 22-amino-acid cyclic lipopeptide with similarity to corpeptin and syringopeptin.

IMPORTANCE Crop rotation and systematic pest management are used to only a limited extent in Greenlandic potato farming.Nonetheless, although plant-pathogenic fungi are present in the soil, the farmers do not experience major plant disease out-breaks. Here, we show that a Greenlandic potato soil is suppressive against Rhizoctonia solani, and we unravel the key biocon-trol components for Pseudomonas fluorescens In5, one of the potent biocontrol bacteria isolated from this Greenlandic suppres-sive soil. Using a combination of molecular genetics, genomics, and microbial imaging mass spectrometry, we show that twocyclic lipopeptides, nunamycin and nunapeptin, are important for the biocontrol activity of P. fluorescens In5 both in vitro andin microcosm assays. Furthermore, we demonstrate that the synthesis of nunamycin is repressed by the oomycete Pythiumaphanidermatum. Overall, our report provides important insight into interkingdom interference between bacteria and fungi/oomycetes.

Received 15 January 2015 Accepted 11 February 2015 Published 17 March 2015

Citation Michelsen CF, Watrous J, Glaring MA, Kersten R, Koyama N, Dorrestein PC, Stougaard P. 2015. Nonribosomal peptides, key biocontrol components for Pseudomonasfluorescens In5, isolated from a Greenlandic suppressive soil. mBio 6(2):e00079-15. doi:10.1128/mBio.00079-15.

Editor E. Peter Greenberg, University of Washington

Copyright © 2015 Michelsen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unportedlicense, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

Address correspondence to Peter Stougaard (queries on microbial genetics work), psg@plen.ku.dk, or Pieter C. Dorrestein (queries on imaging MS), pdorrestein@ucsd.edu.

Agriculture in subarctic Greenland is possible without pesti-cides and with limited crop rotation despite the presence of

plant-pathogenic fungi. For example, potatoes cultivated in asmall single field at the Agricultural Consulting Services in Qaqor-toq for almost 50 years showed no severe incidence of diseasessuch as potato late blight (K. Høegh, personal communication).One plausible explanation is that these Greenlandic soils are dis-ease suppressive, i.e., they contain beneficial microbes that posi-tively affect the growth and health of plants (1, 2). Suppressivesoils have been described from several parts of the world, includ-ing Fusarium wilt-suppressive soils from Chateaurenard (France)

and the Salinas Valley (United States), potato scab-suppressivesoils from Washington (United States), and suppressive sugar beetsoils (the Netherlands) (2, 3). Beneficial microorganisms respon-sible for suppression of fungal growth, the so-called biologicalcontrol agents (BCAs), have been discovered among bacteria ofseveral genera, e.g., Bacillus, Alcaligenes, Burkholderia, Agrobacte-rium, and Pseudomonas (1–4). Of these, fluorescent pseudomon-ads have been investigated in the greatest detail, and antifungalcompounds, including phenazines, pyoluteorin, phloroglucinols,pyrrolnitrin, hydrogen cyanide (HCN), and nonribosomal pep-tides (NRPs), have been identified (5).

RESEARCH ARTICLE crossmark

March/April 2015 Volume 6 Issue 2 e00079-15 ® mbio.asm.org 1

on August 22, 2020 by guest

http://mbio.asm

.org/D

ownloaded from

A Pseudomonas strain with high antifungal activity was previ-ously isolated from a potato field at Inneruulalik, southern Green-land (6). This bacterium, Pseudomonas fluorescens In5, inhibited awide range of pathogenic fungi and oomycetes in vitro in atemperature-dependent manner, i.e., showing higher activities atlower temperatures (6). Previous attempts to identify antifungalcompounds such as phenazines, pyoluteorin, phloroglucinols,and pyrrolnitrin, or the genes encoding such compounds inP. fluorescens In5, were unsuccessful. The only putative antifungalcompound identified was HCN, but genetic analysis indicatedthat an unknown NRP might also be involved in the antifungalactivity of this strain (6, 7). The actual NRP, however, was neveridentified or isolated.

In this study, we unraveled the antifungal mechanism of thedisease-suppressive P. fluorescens In5 strain using a combinationof in vitro agar plate assays, soil microcosm experiments, molecu-lar genetics, genomics, and matrix-assisted laser desorption ion-ization–time of flight (MALDI-TOF) imaging mass spectrometry(IMS) analyses. We show that the main biocontrol ability ofP. fluorescens In5 is mediated by two lipopeptides with distinctfungal inhibition activities depending on the target fungus or oo-mycete.

RESULTSPotato soil from Inneruulalik in southern Greenland is diseasesuppressive. Soil samples were collected from a potato field atInneruulalik in southern Greenland. According to the farmer, po-tatoes were cultivated in the same plot for at least 10 consecutiveyears without any incidence of severe diseases. The disease-suppressive properties of the soil were investigated using in vitrosoil microcosm experiments with seedlings of tomato (a memberof the same plant family as potatoes, Solanaceae) and the plant-pathogenic basidiomycete Rhizoctonia solani Ag3 causing, e.g.,stem rot and tuber black scurf of potatoes. Seedlings planted intothe Inneruulalik soil inoculated with R. solani Ag3 mycelium didnot show any damping-off symptoms, whereas a heat treatment ofthe soil (80°C for 1 h), followed by R. solani Ag3 inoculation, led toseverely affected seedlings (Fig. 1). In addition, the disease-suppressive properties of the heat-treated soil could be almostcompletely recovered by mixing in a small (10%) amount of theuntreated soil (Fig. 1), which suggests that the Inneruulalik soil is

suppressive against R. solani Ag3 and that the suppressive mech-anism is of microbiological origin. Metagenomic DNA was iso-lated from the Inneruulalik suppressive soil, and pyrosequencinggenerated a total of 30,747 high-quality sequences revealing mem-bers of the phyla Proteobacteria, Bacteroidetes, Actinobacteria, andAcidobacteria to be the most abundant bacteria in the soil micro-biome (see Table S1 in the supplemental material). Members ofthe bacterial genera Pseudomonas, Bacillus, Paenibacillus, Strepto-myces, and Burkholderia have previously been described for theirbiocontrol activities (8–11); however, the culture-independentpyrosequencing analysis showed that these bacterial genera con-stituted only a small fraction of the total diversity in the Inneruu-lalik soil (see Table S2). Previously, a potent antifungal pseu-domonad, P. fluorescens In5, was identified from the Inneruulalikpotato soil using culture-based approaches (6); hence, this bio-control bacterium was the subject of the subsequent studies.

Molecular genetics of P. fluorescens In5 reveal an antifungalgenomic island encoding nonribosomal peptides with distinctantifungal activities. To identify genes in P. fluorescens In5 encod-ing antifungal compounds, a Tn5 transposon mutant library wasconstructed and the approximately 1,500 mutants were arrangedin microtiter plate format and screened for loss of mycelial growthinhibition of R. solani Ag3 or Pythium aphanidermatum (an oo-mycete causing leak syndrome of potatoes) in vitro. One mutant,Tn1, failed to inhibit mycelial growth of R. solani Ag3, whereasinhibition of P. aphanidermatum was slightly increased comparedto the level seen with wild-type strain In5 (Fig. 2A; see also Fig. S1in the supplemental material). DNA sequence analysis of the in-sertion site showed that the transposon had inserted into an openreading frame (ORF), nunE, encoding a putative NRP (Fig. 3).Interestingly, a closer examination of the whole-genome sequenceof P. fluorescens In5 revealed that the genomic region around thetransposon insertion site contained a number of ORFs with sim-ilarities to known NRP genes from P. syringae pv. syringae B728a,Cit7, and 642 and from Pseudomonas sp. SHC52 (Fig. 3; see alsoTable S3). The amino acid sequence encoded by nunE and thatencoded by the adjacent nunD ORF showed high similarity to theproducts of the thanamycin synthesis gene cluster from Pseu-domonas sp. SHC52 (60% identity [Id] to ThaA and 76% Id toThaB, respectively) as well as similarity to those of the syringomy-cin synthesis gene cluster (syrE) from P. syringae pv. syringaeB728a, Cit7, and 642 (approximately [approx.] 30% to 40% Id)(Fig. 3; see also Table S3). In addition to nunD and nunE, thenunB1 and nunB2 gene products showed high similarity to thethaC1 and thaC2 gene products from Pseudomonas sp. SHC52(73% and 87% Id, respectively) and to syrB1 and syrB2 gene prod-ucts from P. syringae strains B728a, Cit7, and 642 (approx. 70% to88% Id) (Fig. 3; see also Table S3). SyrB1/ThaC1 and SyrB2/ThaC2 are believed to be involved in selection and activation ofThr-9 and in chlorination of the nonribosomal peptides syringo-mycin and thanamycin, respectively (3, 12, 13). Furthermore, thenunC gene product showed high similarity to ThaD from Pseu-domonas sp. SHC52 (69% Id) and to SyrC from P. syringae strainsB728a, Cit7, and 642 (approx. 69% to 70% Id), which mediate thetransfer of 4-Cl-L-Thr from the NRP-T1 domain to the NRP-T8,9domain as described in the syringomycin synthesis assembly line(12). The peptide encoded by nunB1, nunB2, nunC, nunD, andnunE was denoted “nunamycin” (in Greenlandic, Nuna � Earth/land and Kalaallit Nunaat � Greenland).

Next to the nunamycin biosynthesis gene cluster, other ORFs

FIG 1 Soil microcosm experiment. Presprouted tomato seedlings wereplanted in the Inneruulalik suppressive soil (S), in the Inneruulalik soil heattreated at 80°C (C, conducive), and in heat-treated soil mixed with the sup-pressive soil (9:1 [wt/wt]) (CS). Damping-off symptoms of infected seedlingscaused by R. solani Ag3 were scored visually and expressed as percentages ofdisease incidence (means � standard errors of the means [SEM], n � 4). Theasterisk indicates a statistically significant difference from the suppressive soil(S) (P � 0.05, Tukey’s HSD test).

Michelsen et al.

2 ® mbio.asm.org March/April 2015 Volume 6 Issue 2 e00079-15

on August 22, 2020 by guest

http://mbio.asm

.org/D

ownloaded from

encoding putative NRPs were present. Three ORFs denoted nupA,nupB, and nupC displayed similarity to the syringopeptin biosyn-thesis gene cluster from P. syringae pv. syringae B728a (between36% and 47% Id) (Fig. 3; see also Table S3 in the supplementalmaterial), whereas a corresponding gene cluster was not identifiedin Pseudomonas sp. SHC52 due to a truncated DNA sequencefrom this isolate. The putative peptide encoded by the nupA,nupB, and nupC gene cluster was denoted nunapeptin. In additionto the two nun and nup gene clusters, the antifungal genomicisland in P. fluorescens In5 also contained the genes nupD, pseA topseC (pseA-C), and pseE-F, which in P. syringae have been associ-ated with regulation and export of peptides (Fig. 3; see also Ta-

ble S3) (14). The gene order of the In5 antifungal genomic islandwas identical to that of P. syringae pv. syringae B728a, but thethaC1, thaC2, and thaD genes in Pseudomonas sp. SHC52 werelocated downstream of the major NRP genes thaA and thaB(Fig. 3).

Gene replacement mutants Gr1 (mutated in nunD) and Gr2(mutated in nupB) as well as a double mutant, Tn1Gr2, carryingboth the mutation in Tn1 (mutated in nunE) and that in Gr2, wereconstructed in P. fluorescens In5 (Fig. 3) and analyzed for antifun-gal activities in vitro on agar plates. Mutant Gr1 displayed activitysimilar to that seen with mutant Tn1, i.e., significantly reducedinhibition of R. solani Ag3 mycelial growth compared to that of

FIG 2 (A) Inhibition of R. solani Ag3 (i) or P. aphanidermatum (ii) mycelial growth by P. fluorescens In5 (wt) and the P. fluorescens In5-derived mutant strains,Tn1 (the nunE mutant), Gr1 (the nunD mutant), Gr2 (the nupB mutant), and Tn1 Gr2 (the nunE nupB double mutant). In all cases, the bacteria are seen in thecentral colony on the agar plate surrounded by four identical mycelium plugs. Inhibition of R. solani Ag3 is indicated by white arrowheads, whereas inhibitionof P. aphanidermatum is indicated by black arrowheads. (B) MALDI-TOF imaging mass spectrometry images of nunamycin (NM; red) and nunapeptin (NP;green) produced by P. fluorescens In5 (wt) and the P. fluorescens In5-derived mutant strains, Tn1 and Gr1, cocultured with R. solani Ag3 (i) or P. aphanidermatum(ii). (C) Table summarizing production of nunamycin (NM) and nunapeptin (NP) by the P. fluorescens In5 wt and mutant strains Tn1, Gr1, Gr2, and Tn1Gr2as evaluated by MALDI-TOF IMS and/or MS analysis. �, production of peptide; �, no production of peptide.

FIG 3 Map of the antifungal genome cluster of P. fluorescens In5 (A) and closest relatives Pseudomonas sp. SHC52 (GenBank accession no. HQ888764) (B) andP. syringae pv. syringae B728a (GenBank accession number NC_007005) (C). Open triangles show the gene replacement mutations corresponding to Gr1 andGr2; the filled triangle shows the transposon mutation corresponding to Tn1. Grey arrow boxes with a boldface capital E denote genes involved in export; openarrow boxes with a boldface capital R denote genes involved in regulation.

Antifungal Peptides from Pseudomonas

March/April 2015 Volume 6 Issue 2 e00079-15 ® mbio.asm.org 3

on August 22, 2020 by guest

http://mbio.asm

.org/D

ownloaded from

wild-type strain In5, whereas inhibition of P. aphanidermatumwas slightly increased (Fig. 2A; see also Fig. S1 in the supplementalmaterial). The biocontrol activity of the nunE mutant, Tn1, wasfurther studied in soil microcosm experiments, and results re-vealed a substantially reduced ability to protect tomato seedlingsagainst R. solani Ag3 compared to the wild-type In5 strain (Fig. 4).The nupB mutant, Gr2, had completely lost the ability to inhibitP. aphanidermatum mycelial growth, whereas the same low levelof inhibition against R. solani Ag3 as that seen with the mutantsGr1 and Tn1 was observed (Fig. 2A; see also Fig. S1), indicatingthat, in contrast to the nun genes, nupB plays a role in biocontrolof P. aphanidermatum. Furthermore, the Tn1Gr2 double mutantshowed virtually no in vitro inhibition activity against R. solaniAg3 or P. aphanidermatum (Fig. 2A; see also Fig. S1).

Identification of nonribosomal peptides produced by P. fluo-rescens In5 using MALDI-TOF imaging mass spectrometry. Inorder to bypass laborious and time-consuming conventionalpeptide purification methods, we used matrix-assisted laser des-orption–ionization time of flight (MALDI-TOF) imaging massspectrometry (IMS) to identify the antifungal peptides encodedby the antifungal genomic island in P. fluorescens In5. Ions ofinterest were selected by superimposing the false-colored ion dis-tribution map from the IMS analysis onto an optical image of thebacterium-fungus interaction and searching for ions with a strongpresence within the area of fungal inhibition (Fig. 2B and C). Incocultures of the P. fluorescens In5 wild type and R. solani Ag3, ionswith a mass of m/z 1,138 and a family of m/z 2,023 to 2,075, withthe highest intensity observed at m/z 2,060, were detected, whereas

mutant strains Tn1 and Gr1 produced only the m/z 2,060 ion(Fig. 2B and C). These results showed that the m/z 1,138 ion cor-responded to the nunamycin peptide and indicated that the m/z2,060 ion corresponded to the nunapeptin peptide. MALDI-TOFmass spectrometry (MS) analysis of the P. fluorescens In5 wild typeand the mutant strains further confirmed the production of bothnunamycin and nunapeptin by the In5 wild-type strain, whereasthe mutant strains, Tn1 and Gr1, produced only nunapeptin and,notably, the mutant strains, Gr2 and Tn1Gr2, showed no produc-tion of either nunamycin or nunapeptin (Fig. 2C; see also Fig. S2in the supplemental material).

Surprisingly, we noticed in the MALDI-TOF IMS analysisthat cocultures with P. fluorescens In5 and P. aphanidermatumshowed the presence only of nunapeptin (i.e., m/z 2,060 ion) andnot of nunamycin (i.e., m/z 1,138 ion) (Fig. 2B), indicating thatP. aphanidermatum inhibited the synthesis of nunamycin in strainIn5. Corroborating the results from coculture experiments, thecrude peptide extract from P. fluorescens In5 containing both nu-namycin and nunapeptin inhibited mycelial growth of both R. so-lani Ag3 and P. aphanidermatum, whereas the purified nunamycinpeptide inhibited R. solani Ag3 but not P. aphanidermatum, andthe purified nunapeptin in contrast showed inhibition ofP. aphanidermatum but not of R. solani Ag3 (Fig. 5).

The expression of nunamycin and nunapeptin was analyzedover time using MALDI-TOF MS. The two ions m/z 1,138 and2,060, corresponding to nunamycin and nunapeptin, respectively,first appeared 12 h after inoculation of P. fluorescens In5, with amaximum mass signal observed at 48 to 72 h (Fig. 6). In the twonun mutants, Tn1 and Gr1, the m/z 1,138 ion was absent (Fig. 6).After 7 days, the m/z 1,138 ion was no longer visible, whereas them/z 2,060 ion was present but at a lower intensity (Fig. 6).

Molecular structures of nunamycin and nunapeptin revealtwo cyclic lipopeptides. The molecular structures of nunamycinand nunapeptin are proposed on the basis of a combination ofnuclear magnetic resonance (NMR)- and mass spectrometry(MS)-based methods (Fig. 7). For nunamycin, initial analysis ofthe parent peak at m/z 1,138.54 by high-resolution mass spec-trometry indicated the presence of a single chlorine atom within

FIG 4 Soil microcosm experiment. The microcosm experiment was carriedout with soil containing R. solani Ag3 (R) that was inoculated with P. fluore-scens strain In5 wt and R. solani Ag3 (In5-R) or with mutant strain Tn1 and R.solani Ag3 (Tn1-R) and maintained at 15°C (A) or 20°C (B); the results werecompared to those seen with untreated control soil (column C), soil inoculatedwith P. fluorescens In5 wt (In5) or mutant strain Tn1 (Tn1). Damping-offsymptoms of infected seedlings caused by R. solani Ag3 were scored visuallyand expressed as percentages of disease incidence (means � SEM, n � 4).Asterisks indicate statistically significant differences from the control treat-ment (column C) (P � 0.05, Tukey’s HSD test).

FIG 5 Inhibition of R. solani Ag3 (i) or P. aphanidermatum (ii) mycelialgrowth by 10 �g crude peptide extract derived from the P. fluorescens In5 wildtype containing both the nunamycin and nunapeptin peptides (NM�NP);10 �g of the purified nunamycin (NM) or nunapeptin (NP) peptides wasspotted on sterile pieces of filter paper in the center of the agar plate sur-rounded by four identical mycelium plugs. Inhibition of R. solani Ag3 is indi-cated by white arrowheads, whereas inhibition of P. aphanidermatum is indi-cated by black arrowheads.

Michelsen et al.

4 ® mbio.asm.org March/April 2015 Volume 6 Issue 2 e00079-15

on August 22, 2020 by guest

http://mbio.asm

.org/D

ownloaded from

the structure due to a 24% increase in the M �2 peak due to the37Cl isotope of chlorine. Analysis of the gene cluster also showedthe presence of a chlorinase enzyme adjacent to an adenylationdomain capable of loading a 4-Cl threonine residue (see Fig. S3 inthe supplemental material). Analysis of the nunamycin gene clus-ter and comparisons to the syringomycin and thanamycin geneclusters indicated a missing A domain in the modular organiza-tion of the nunamycin NRP (i.e., in module 5 as shown in Fig. S3);at the same time, further annotation of NMR, tandem MS (MS/MS), and MS/MS/MS data revealed that nunamycin is a cycliclipopeptide, consistent with the presence of the 9 amino acid res-idues Ser-Dab-Gly-Hse-Dab-Thr-Thr-(3-OH)Asp-(4-Cl-Thr)(see the Fig. 7 legend for definitions of abbreviations) attached toa 3-hydroxy fatty acid tail (Fig. 7; see also Fig. S3).

Analysis of the nunapeptin gene cluster combined with MS/MSanalysis revealed the presence of a 22-amino-acid cyclic lipopep-

tide, i.e., Dhb-Pro-Ala-Ala-Ala-Val-Ala-Dhb-Ser-Val-Ile-Dha-Ala-Val-Ala-Val-Dhb-Thr-Ala-Dab-Ser-Ile, with strong similarityto corpeptin and syringopeptin (Fig. 7; see also Fig. S4 in thesupplemental material). By high-resolution mass spectrometry,the primary ion at m/z 2,045.20 gave a clear sequence tag for16 amino acid residues with the sequence Pro-Ala-Ala-Ala-Val-Ala-Dhb-Ser-Val-Ile-Dha-Ala-Val-Ala-Val-Thr. In addition, twolarger fragments at m/z 254.175 and m/z 473.272 appeared in themass spectrum. The molecular formula of the m/z 254.175 frag-ment correlated with a Dhb residue attached to 3-OH dodecanoicacid (Fig. 7; see also Fig. S4), which is similar to the structure ofsyringopeptin and corpeptin (15). The molecular formula for them/z 473.272 fragment was consistent with a Thr-Ala-Dab-Ser-Ilecyclic peptide moiety (Fig. 7; see also Fig. S4), which is identical tothe cyclic tail of corpeptin (15). The results of two-dimensional(2D) homo- and heteronuclear NMR experiments supported themass spectral interpretation (see Table S4). In addition to theprimary nunapeptin, three analogs were observed in the massspectrum of In5 (m/z 2,023.22, m/z 2,037.24, and m/z 2,075.22)(see Fig. S2). Tandem MS analysis of these analogs showed iden-tical mass losses for the lipid tail (m/z 254.175) as well as for theterminal cyclic peptide (m/z 473.272).

DISCUSSION

In certain subarctic fields in Greenland, it is possible to cultivatepotatoes with limited crop rotation and no use of pesticides, eventhough plant-pathogenic fungi are present in the soils. Using soilmicrocosms, we showed that a potato soil from Inneruulalik insouthern Greenland was able to suppress the growth of the plant-pathogenic fungus Rhizoctonia solani Ag3. Such suppressive soilsexist worldwide (2), but this report is the first to describe a disease-suppressive soil from a subarctic region. Antifungal microorgan-isms from suppressive soils have been shown to comprise severalbacterial genera (2, 8–11), and an analysis of the Inneruulalik soilby 16S rRNA gene pyrosequencing identified the presence of or-ganisms belonging to several of these genera, i.e., Pseudomonas,Bacillus, Paenibacillus, Streptomyces, and Burkholderia. However,the frequency of organisms of these genera constituted in total less

FIG 6 Time course experiments with the P. fluorescens In5 wild type (A) andthe mutant strains, P. fluorescens Tn1 (B) and P. fluorescens Gr1 (C), culturedon solid 1/5 PDA. Production of nunamycin (NM) (i.e., m/z 1,138) and mem-bers of the nunapeptin family (NP) (i.e., m/z 2,060) by the bacterial strains wasmeasured using MALDI-TOF dried-droplet mass spectrometry at 6 h, 12 h,24 h, 36 h, 48 h, 72 h, 96 h, and 7 days (d) of incubation as indicated by each lineon the y axis. Mass spectra are displayed as stacked heat maps. Each numberedrow is a single mass spectrum with peak intensities represented as a color scale,where low-intensity signals are closer to white and higher-intensity signals arecloser to black as shown in the panel to the right of the spectrum. arb.u.,arbitrary units.

FIG 7 The proposed structures of nunamycin from P. fluorescens In5 and the analogous peptides thanamycin, cormycin A, and syringomycin (A) and ofnunapeptin from P. fluorescens In5 and the analogous peptides corpeptin A and syringopeptin (B). The abbreviations are as follows: (3-OH)Asp,3-hydroxyaspartic acid; (4-Cl)Thr, 4-chlorothreonine; Dab, 2,4-diaminobutyric acid; Dhb, dehydrobutyrine; Dha, dehydroalanine; Orn, ornithine; Hse, ho-moserine. Consensus amino acids are denoted in gray-lined boxes.

Antifungal Peptides from Pseudomonas

March/April 2015 Volume 6 Issue 2 e00079-15 ® mbio.asm.org 5

on August 22, 2020 by guest

http://mbio.asm

.org/D

ownloaded from

than 1% of all phylotypes, which led us to the assumption that thebacteria present in the disease-suppressive Inneruulalik soil mustbe potent inhibitors of fungal growth. An effective biocontrol bac-terium, P. fluorescens In5, was previously isolated from the Inne-ruulalik soil and was found to encode genes for the synthesis ofantifungal compounds, including hydrogen cyanide and ahitherto-uncharacterized nonribosomal peptide (6, 7).

Nonribosomal peptides isolated from several pseudomonadsusing conventional methods such as extraction of active com-pounds in organic solvents followed by high-pressure liquid chro-matography (HPLC) fractionation and structure resolution byNMR and MS have been described previously (16–18). In thisstudy, we demonstrated that molecular genetics combined withgenomics and MALDI-TOF IMS may facilitate the process ofidentification and purification of NRPs and in addition assist inverifying the presence of the desired compounds within the dif-ferent extraction and purification steps. Furthermore, this ap-proach facilitated expression studies of the individual NRPs inlive bacterial-fungal cocultures both spatially and temporally.MALDI-TOF IMS analysis of P. fluorescens In5 revealed the pres-ence of two antifungal NRPs in the inhibition zone of R. solaniAG3 and P. aphanidermatum, respectively. These NRPs, denotednunamycin and nunapeptin, showed structural resemblance tosyringomycin and syringopeptin from P. syringae and to cor-mycin A and corpeptin A from P. corrugata (15) as well as tothanamycin from Pseudomonas sp. SH-C52 (19). Nunamycinwas found to be a chlorinated cyclic lipopeptide composed of9 amino acid residues, Ser-Dab-Gly-Hse-Dab-Thr-Thr-(3-OH)Asp-(4-Cl-Thr), with a molecular mass of 1,137.54 Da,while nunapeptin was produced as a family of cyclic lipopep-tides of 22 amino acid residues, Dhb-Pro-Ala-Ala-Ala-Val-Ala-Dhb-Ser-Val-Ile-Dha-Ala-Val-Ala-Val-Dhb-Thr-Ala-Dab-Ser-Ile,with molecular masses ranging from 2,023 to 2,075 Da. The struc-tures of nunamycin and nunapeptin contained some uncommonamino acids, such as (3-OH)Asp (3-hydroxyaspartic acid), (4-Cl)Thr (4-chlorothreonine), Dab (2,4-diaminobutyric acid), Dhb(dehydrobutyrine), and Dha (dehydroalanine). However, theseamino acids are present in a range of nonribosomal peptides, in-cluding antimicrobial peptides of the syringomycin, tolaasin, andsyringopeptin families (15).

While the biosynthesis of nunapeptin is catalyzed by threeNRPs (NupA, NupB, and NupC) in a collinear manner, the bio-synthesis of nunamycin, catalyzed by the three NRPs, NunB1/NunB2, NunD, and NunE, does not respect the collinearity rule.For example, a split-module phenomenon was found in the ninthmodule in the nunamycin NRP, NunE; the C and T domains werepresent, but an A domain was not, which instead was present inNunB1, as also described for the syringomycin synthase (15). Fur-thermore, an additional A domain was lacking in module 5 of thenunamycin NRP NunD. However, chemical analysis showed thatDab was present as the fifth amino acid in the nunamycin peptidechain; thus, we speculate that the A domain in module 2 couldfunction twice. Such module shuffling has been documented be-fore in NRP-catalyzed peptide synthesis (20).

Transposon and gene replacement mutants, i.e., Tn1, Gr1,Gr2, and Tn1Gr2, constructed in P. fluorescens In5 confirmed thatnunamycin and nunapeptin are key components for the biocon-trol ability observed by this strain. However, the two peptidesshowed distinct inhibition activities. Nunamycin was found toplay a role primarily in R. solani AG3 mycelial growth inhibition,

whereas nunapeptin was essential for P. aphanidermatum inhibi-tion. However, when a nunapeptin NRP knockout mutant (Gr2,mutated in nupB) was analyzed in coculture, this mutant was de-fective in growth inhibition of both P. aphanidermatum and R. so-lani Ag3, although the nunamycin biosynthesis was intact. Acloser examination of the nup operon showed the presence of sixgenes, nupD, pseA-C, and pseE-F, possibly involved in the efflux ofNRPs (14, 21). Thus, it is likely that the knockout of the nupB genein P. fluorescens In5 negatively influences not only the synthesis ofnunapeptin but also the efflux system located downstream ofthe nup genes. If expression of the pse genes is affected, thiscould inhibit efflux of both the nunapeptin and nunamycin andconsequently also affect the inhibition activity against bothP. aphanidermatum and R. solani Ag3. This hypothesis was sup-ported by MALDI-TOF MS analysis, which showed no produc-tion of nunapeptin or nunamycin by this mutant strain.

MALDI-TOF IMS enabled us to study the expression of nuna-mycin and nunapeptin simultaneously and in relation to interac-tions between P. fluorescens In5 and R. solani Ag3 or P. aphanider-matum. Nunamycin and nunapeptin were both expressed incocultures of P. fluorescens In5 with R. solani Ag3. However, onlynunapeptin was present in cocultures with P. aphanidermatum,suggesting that the synthesis of nunamycin in P. fluorescens In5was inhibited by P. aphanidermatum. Regulation of lipopeptidesand other secondary metabolites in pseudomonads by other or-ganisms has been reported in several cases. For example, the pro-duction of secondary metabolites in P. aeruginosa was found to beaffected by a halogenated furanone compound produced by theAustralian macroalga Delisea pulchra (22), and sugar beet extractswere shown to affect lipopeptide synthesis in Pseudomonas sp.DSS73 (23). The exact mechanism by which P. aphanidermatuminhibited the biosynthesis of nunamycin in P. fluorescens In5 inthis study, however, still needs to be elucidated.

MALDI-TOF MS analysis similarly permitted a study of theexpression of nunamycin and nunapeptin by P. fluorescens In5wild-type and mutant strains and allowed a time course analysis ofnunamycin and nunapeptin synthesis by P. fluorescens In5. Nuna-mycin and nunapeptin were first detected 12 h after inoculation,with signals for the peptides reaching a maximum after 48 to 72 hof incubation and subsequently decreasing after 7 days of incuba-tion. This expression profile is different from other lipopeptidesynthesis profiles. For example, in P. fluorescens strain SS101, mas-setolide A biosynthesis initiates between 12 h and 16 h of incuba-tion and peaks after 24 h of incubation (24). In Bacillus amyloliq-uefaciens, synthesis of surfactin and fengycin displayed maximalproduction after 10 to 40 h and decreased after 60 h, whereasbacillomycin D showed maximal intensity after 40 and 60 h (25).The transient production of nunamycin, however, has previouslybeen observed with the analogous molecule thanamycin fromPseudomonas sp. SH-C52 (19).

The results presented in this report demonstrate the powerfulapproach of combining in vitro inhibition experiments with mo-lecular genetics, genomics, and MALDI-TOF IMS analyses. Usingthis combination of tools, we were able to unravel the key biocon-trol factors of a Greenlandic bacterium, P. fluorescens In5, isolatedfrom a disease-suppressive potato soil in subarctic Greenland, toelucidate the structure and function of two antifungal lipopep-tides, and to document that the effective biocontrol ability ofP. fluorescens In5 requires the synthesis of both peptides.

Michelsen et al.

6 ® mbio.asm.org March/April 2015 Volume 6 Issue 2 e00079-15

on August 22, 2020 by guest

http://mbio.asm

.org/D

ownloaded from

MATERIALS AND METHODSStrains and culture conditions. Bacterial strains (Table 1) were routinelygrown in liquid or on solid (1.5% agar) Luria-Bertani (LB) medium at28°C (Pseudomonas strains) or at 37°C (Escherichia coli [for transposonmutagenesis]). When required, the medium was supplemented withthe following antibiotic(s): kanamycin (50 �g·ml�1), ampicillin(50 �g·ml�1), or gentamicin (10 �g·ml�1). The basidiomycete R. solaniAg3 and the oomycete P. aphanidermatum (Table 1) were used as targetpathogens in in vitro inhibition assays and soil microcosm experiments.The fungus or oomycete was maintained on standard potato dextrose agar(PDA; BD/Difco, USA) medium at 20°C.

Soil sampling, DNA isolation, and pyrosequencing analysis of theInneruulalik potato soil. For details on soil sampling, DNA isolation, andpyrosequencing analysis of the Inneruulalik potato soil, see Materials andMethods in the supplemental material.

Soil microcosm assays. The Inneruulalik soil was sieved (0.5-cm-pore-size mesh) to remove plant debris and small stones. Tomato seeds(cv. Marmande Super) were presprouted for 4 days at 20°C in petri disheson wetted filter paper.

For determination of soil suppressiveness, four presprouted tomatoseedlings were placed randomly in petri dishes filled with 40 g of soil withan initial moisture content of 14.8% (vol/wt). The various soil treatmentswere suppressive soil (S), suppressive soil heat-treated at 80°C in an ovenfor 1 h (C), and heat-treated soil mixed with 10% (wt/wt) suppressive soil(CS). R. solani Ag3 was introduced into the soil by transferring one quar-ter of a mycelium agar plug (6-mm diameter) of a 1-week-old PDA plateto each corner of a petri dish. Plates were incubated in a growth chamberat 15°C using a 16-h:8-h light:dark cycle and watered after 1 week ofincubation with 5 ml of standard Hoagland solution (macronutrientsonly). Four replicates (i.e., plates) were made for each soil treatment, andthe experiment was repeated twice. After 12 days of incubation, the per-centage of infected tomato seedlings showing damping-off symptoms wasscored.

In order to determine the R. solani Ag3-suppressive activity by theP. fluorescens In5 wild type and the P. fluorescens Tn1 mutant strain, soilwas initially heat-treated at 80°C in an oven for 1 h. Four presproutedtomato seedlings were placed randomly in petri dishes filled with 40 g ofthe heat-treated soil with or without addition (2 ml/100 g) of overnight(O/N) bacterial cultures washed twice with LB and adjusted to an opticaldensity at 600 nm (OD600) of 2. R. solani Ag3 was introduced into the soilas described above. The various soil treatments were control soil, tomatoseedlings only (C), soil inoculated with the P. fluorescens In5 wild type(In5), soil inoculated with mutant strain P. fluorescens Tn1 (Tn1), soilinfected with R. solani Ag3 (R), soil inoculated with the P. fluorescens In5

wild type and infected with R. solani Ag3 (In5-R), and soil inoculated withmutant strain P. fluorescens Tn1 and infected with R. solani Ag3 (Tn1-R).Plates were incubated in a growth chamber at 15°C or 20°C with a 16-h:8-h light:dark cycle. Plates were watered as described above. Four repli-cates (i.e., plates) were made for each soil treatment, and the experimentwas repeated twice. After 10 and 6 days of incubation at 15°C and 20°C,respectively, the percentage of infected tomato seedlings showingdamping-off symptoms was scored.

Screening of the transposon mutant library. Transposon mutagene-sis of P. fluorescens In5 was performed using a mobilizable plasposon,pTnModOKm (26), as previously described (27). Strain In5 transconju-gants with transposons integrated into the chromosome were selected onLB agar plates supplemented with kanamycin and ampicillin. Approxi-mately 1,500 mutants were screened for lack of antifungal activity. Mu-tants were grown O/N in F96 Microwell plate (Nunc) (in 150 �l LB withampicillin [50 �g·ml�1 per well] and kanamycin [50 �g·ml�1 per well]).Overnight cultures (1.5 �l) were transferred by a multichannel pipette toOmniTrays (Nunc) containing PDA at a strength of 1/5 (1/5 PDA) previ-ously inoculated with a suspension of blended fungal hyphae of R. solaniAg3 or P. aphanidermatum. Transposon rescue of selected mutants usingXhoI or SacII was performed as previously described (6).

Homologous recombination. Gene knockout by homologous recom-bination in the m/z 1,138 and m/z 2,060 NRP gene clusters was carried outusing a gene replacement vector, pEX100T (28). Approximately 2,000 to2,300 bp of the m/z 1,138 and m/z 2,060 NRP gene clusters containing oneor two SalI sites were amplified with primers NRPS13a (5=-CGCATAACCGCACACTG=-3) and NRPS13b (5=-CTGGAACAAGTCGGTCGC=-3)and primers 2 F (5=-TGTCGAGGCTTGCGCCAAC=-3) and 2 R (5=-CGCGGAAGCGGTGATCCT=-3), respectively. The m/z 1,138 and m/z 2,060NRP gene fragments were amplified using Phusion blunt-end polymerase(annealing temperature, 63°C), according to the manufacturer’s instruc-tions. The blunt-end-amplified fragments were ligated into SmaI-digested pEX100T. The pEX100T vectors with insertions were digestedwith SalI, and a SalI-digested gentamicin resistance (Gmr) gene and apromoter from plasmid Tn7gfp2 (29) were inserted into the NRP geneclusters. The pEX100T vectors with NRP fragments disrupted by the Gmr

gene were transformed into electrocompetent P. fluorescens In5 cells to-gether with the pUX-BF13 helper plasmid as previously described (30).Strain In5 transformants with a site-directed knockout in one of the twoNRP gene clusters were selected on LB agar plates supplemented withampicillin and gentamicin.

Fungus/oomycete inhibition assays on agar plates. Antifungal activ-ity of the different strains and the purified nunamycin and nunapeptinwas tested on 1/5 PDA against R. solani Ag3 or P. aphanidermatum. Ten

TABLE 1 Bacteria, fungi, and plasmids used in this study

Plasmid or bacterial or fungal strain Relevant genotype and (or) characteristic(s)a Reference

PlasmidsppiKmLacZ Kmr-lacZ cassette; pTnMod-based; mobilizable 26pRK2013 Self-transmissible helper plasmid; Kmr 27pEX100T oriT, bla, sacB 28

BacteriaEscherichia coli DH5� endA1 hsdR17 supE44 thi-1 recA1 U169 deoR 34Pseudomonas fluorescens In5 WT 6Pseudomonas fluorescens In5-Tn1 �nunE 6Pseudomonas fluorescens In5-Gr1 �nunD This studyPseudomonas fluorescens In5-Gr2 �nupB This studyPseudomonas fluorescens In5-Tn1Gr2 �nunE �nupB This study

Fungus or oomyceteRhizoctonia solani Ag3 Basidiomycete 6Pythium aphanidermatum Oomycete 6

a Km, kanamycin; WT, wild type.

Antifungal Peptides from Pseudomonas

March/April 2015 Volume 6 Issue 2 e00079-15 ® mbio.asm.org 7

on August 22, 2020 by guest

http://mbio.asm

.org/D

ownloaded from

microliters of O/N bacterial cultures or 10 �g of the crude peptide extractor purified nunamycin and nunapeptin peptides on sterile filter paperswas inoculated in the center of the agar plates. Sterile MilliQ purified H2Oon filter paper was used as a control for the peptide extract and purifiedpeptides. Four fungus/oomycete mycelium plugs were placed 2 cm fromthe center of the plate around the colony or filter paper. Plates were madein triplicate and incubated at 15°C (P. aphanidermatum) or 20°C (R. solaniAg3). Following 48 to 240 h of coculturing, depending on the fungus/oomycete strain and incubation temperature used, all plates were visuallyinspected and the inhibitory effect of the bacterial strains was evaluated aspercent inhibition of radial growth (PIRG) as previously described (31).

MALDI-TOF imaging mass spectrometry analysis. P. fluorescens In5or mutant strain Tn1 or Gr1 was cocultured with R. solani Ag3 orP. aphanidermatum on thin 1/5 PDA plates (i.e., 10 ml of media in astandard petri dish, resulting in a thickness of medium of about 1.5 mm).Once the desired time point for growth was reached, areas of agar mediacontaining the microbial colonies were excised from the petri dish andtransferred to the MALDI stainless steel target plate. The subsequent ma-trix application was performed using a 53-�m-pore-size test sieve; drymatrix was deposited evenly across the sample surface until the desiredmatrix thickness was achieved. The sample was dried O/N followed byMALDI-TOF IMS analysis using a Bruker Microflex mass spectrometer.Data were collected from 50 to 3,000 m/z at 80-Hz laser frequency at aspatial resolution of 400 �m using a random-walk shot pattern. Data wereanalyzed using the Compass 1.2 software suite (FlexImaging 2.0, FlexCon-trol 3.0, and FlexAnalysis 3.0; Bruker Daltonics) as previously described(32). The resulting mass spectrum was binned at 0.5-m/z increments andmanually inspected for masses of interest. Masses were visualized usingfalse coloring on an optical image of the original sample plate.

Peptide extraction. Plates with 1/5 PDA medium were inoculatedwith P. fluorescens In5. After incubation for 48 h at room temperature, theagar was sliced into small pieces and extracted with acidified deionizedwater (pH 2) for 1 h at 28°C and 225 rpm in a 2.8-liter Erlenmeyer flaskfollowed by addition of n-butanol and incubation for 18 h at 28°C. Then-butanol extract was separated from agar by cheesecloth filtration andsubsequently from cell debris by centrifugation (6,440 � g, 10 min).n-Butanol was removed with a rotovaporator (Buechi R-200). The crudepeptide extract was resuspended in 1 ml methanol and analyzed byMALDI-TOF mass spectrometry (MS). The resuspended extract was cen-trifuged for 2 min at 16,000 � g, and the supernatant was placed on amethanol-equilibrated Sephadex LH20 column (30 cm length). The pep-tide extract was separated with a methanol mobile phase at a flow rate of0.4 ml/min. Fractions of 7.5 ml were collected, lyophilized, and resus-pended in 200 �l methanol. Gel filtration fractions were analyzed byMALDI-TOF MS for peptide content. Fractions of the peptide-containinggel filtration were combined and concentrated by lyophilization. The pep-tides were further purified by reversed-phase high-pressure liquid chro-matography (HPLC) using an Agilent 1260 system equipped with a Phe-nomenex Kinetex C18 column (4.6 mm by 15 cm). The peptides wereeluted from the column using a gradient of 10% acetonitrile containing0.1% formic acid:90% water containing 0.1% formic acid to 90% aceto-nitrile containing 0.1% formic acid:10% water containing 0.1% formicacid over 45 min at a flow rate of 1.0 ml/min. Peaks for nunamycin andnunapeptin were collected manually (24.6 min for nunamycin and mul-tiple peaks between 35 and 40 min for the nunapeptin family of com-pounds), after which they were lyophilized and stored at �80°C.

NMR and tandem MS analysis. About 1 mg of nunapeptin at m/z2,023 to 2,075 and about 100 �g of nunamycin at m/z 1,138 were dissolvedin 40 �l of deuterated methanol (CD3OD) for NMR data acquisition.NMR spectra were recorded on a Bruker Avance iii 600-Mhz spectrometerwith a 1.7-mm-diameter Microbiology CryoProbe at 298 K, with standardpulse sequences provided by Bruker. Structures were confirmed by anal-ysis of 1D proton, 2D Cosy double-quantum filter (Dqf-Cosy), 2D 1H-13C heteronuclear single-quantum coherence (HSQC), and 2D 1H-13Cheteronuclear multiple-bond correlation (HMBC) spectra. 2D 1H-13C

HMBC spectra were recorded with delays corresponding to a 2j or 3j H-Ccoupling constant of 8 Hz and a 1j H-C coupling constant of 145 Hz. 2D1H-13C multiplicity-edited HSQC spectra were recorded, with delayscorresponding to a 1j H-C coupling constant of 140 Hz. Spectra werereferenced to the methanol methyl resonance at 3.31 ppm.

Tandem mass spectrometry experiments were performed on aThermo LTQ fault-tolerant (FT) mass spectrometer (Thermo ElectronCorp., Bremen, Germany) equipped with a Diversa Nanomate electros-pray source. Aliquots from solutions of purified nunapeptin and nuna-mycin compounds were diluted in acetonitrile:0.1% formic acid (70:30[vol/vol]). Electrospray parameters included a spray voltage of 1.40 kV,heated inlet temperature of 200°C, nebulizing gas pressure of 40 lb/in2 N2,collision-induced dissociation energy of 25 V, and collision time of500 ms. The instrument was operated in FT mode (peak resolution, 50 k)for detection of parent (MS) and daughter (MS/MS) ion peaks.

MALDI-TOF mass spectrometry. MALDI-TOF MS was used to de-tect target peptides in the crude peptide extract and fractions from gelfiltration and HPLC as well as peptide production over time in the timecourse experiment. The sample was mixed 1:1 with a saturated solution ofUniversal MALDI matrix–70% acetonitrile containing 0.1% trifluoro-acetic acid (TFA) and spotted on a Bruker MSP 96 anchor plate. The targetplate containing the samples was dried and inserted into a MicroflexBruker Daltonics mass spectrometer. External calibration was done asdescribed for MALDI-TOF IMS. Mass spectra were obtained with theFlexControl method as used for MALDI-TOF IMS and a single-spot ac-quisition of 200 shots. Single-spot MALDI-TOF MS data were analyzed byFlexAnalysis software.

Time course experiment of peptide production. Cultures of theP. fluorescens In5 wild type and the mutant strains, P. fluorescens Tn1 andGr1, were inoculated on solid 1/5 PDA at 20°C and sampled at specifictime points. Samples for dry-droplet analysis of peptide production (m/z1,138 and 2,060) by MALDI-MS were taken after 6, 12, 24, 36, 48, 72, and96 h and after 7 days. A scrape of bacterial colonies was dissolved in 100 �lof sterile deionized water. The bacterial suspension was mixed 1:1 with asaturated solution of Universal MALDI matrix in 70% acetonitrile con-taining 0.1% TFA and spotted on a Bruker MSP 96 anchor plate. MALDIMS analysis was carried out as described above.

Statistical analysis. Tukey’s honestly significant difference (HSD) testwas used in conjunction with analysis of variance (ANOVA) in order toevaluate the data from the in vitro inhibition and microcosm experimentsto identify means with significant differences. The tests were carried outwith R version 2.10.0 (33).

SUPPLEMENTAL MATERIALSupplemental material for this article may be found at http://mbio.asm.org/lookup/suppl/doi:10.1128/mBio.00079-15/-/DCSupplemental.

Text S1, DOC file, 0.01 MB.Figure S1, PDF file, 1 MB.Figure S2, PDF file, 0.2 MB.Figure S3, PDF file, 0.2 MB.Figure S4, PDF file, 0.3 MB.Table S1, PDF file, 0.2 MB.Table S2, PDF file, 0.1 MB.Table S3, PDF file, 0.1 MB.Table S4, PDF file, 0.1 MB.

ACKNOWLEDGMENTS

Referring to the Convention on Biological Diversity, we thank the gov-ernment of Greenland and the Greenlandic farmers for permission tosample soil and bacteria in southern Greenland.

We thank Laura Sanchez for consultations on the NMR analysis.This work was funded in part by the Commission for Scientific Re-

search in Greenland and the Augustinus Foundation and NIHGM097509, NIHAI095125, and S10RR029121.

Michelsen et al.

8 ® mbio.asm.org March/April 2015 Volume 6 Issue 2 e00079-15

on August 22, 2020 by guest

http://mbio.asm

.org/D

ownloaded from

REFERENCES1. Raaijmakers JM, Paulitz TC, Steinberg C, Alabouvette C, Moënne-

Loccoz Y. 2009. The rhizosphere: a playground and battlefield for soil-borne pathogens and beneficial microorganisms. Plant Soil 321:341–361.http://dx.doi.org/10.1007/s11104-008-9568-6.

2. Weller DM, Raaijmakers JM, Gardener BB, Thomashow LS. 2002.Microbial populations responsible for specific soil suppressiveness toplant pathogens. Annu Rev Phytopathol 40:309 –348. http://dx.doi.org/10.1146/annurev.phyto.40.030402.110010.

3. Mendes R, Kruijt M, de Bruijn I, Dekkers E, Mendes R, Kruijt M, deBruijn I, Dekkers E, van der Voort M, Schneider JH, Piceno YM,DeSantis TZ, Andersen GL, Bakker PA, Raaijmakers JM. 2011. Deci-phering the rhizosphere microbiome for disease-suppressive bacteria. Sci-ence 332:1097–1100. http://dx.doi.org/10.1126/science.1203980.

4. Yuen GY, Schroth MN. 1986. Inhibition of Fusarium oxysporum f. sp.dianthi by iron competition with an Alcaligenes sp. Phytopathology 76:171–176. http://dx.doi.org/10.1094/Phyto-76-171.

5. Haas D, Défago G. 2005. Biological control of soil-borne pathogens byfluorescent pseudomonads. Nat Rev Microbiol 3:307–319. http://dx.doi.org/10.1038/nrmicro1129.

6. Michelsen CF, Stougaard P. 2011. A novel antifungal Pseudomonas fluo-rescens isolated from potato soils in Greenland. Curr Microbiol 62:1185–1192. http://dx.doi.org/10.1007/s00284-010-9846-4.

7. Michelsen CF, Stougaard P. 2012. Hydrogen cyanide synthesis and anti-fungal activity of the biocontrol strain Pseudomonas fluorescens In5 fromGreenland is highly dependent on growth medium. Can J Microbiol 58:381–390. http://dx.doi.org/10.1139/w2012-004.

8. Lemanceau P, Alabouvette C. 1991. Biological control of Fusarium dis-eases by fluorescent Pseudomonas and nonpathogenic Fusarium. CropProtect 10:279 –286. http://dx.doi.org/10.1016/0261-2194(91)90006-D.

9. Ongena M, Jacques P. 2008. Bacillus lipopeptides: versatile weapons forplant disease biocontrol. Trends Microbiol 16:115–125. http://dx.doi.org/10.1016/j.tim.2007.12.009.

10. Son SH, Khan Z, Kim SG, Kim YH. 2009. Plant growth-promotingrhizobacteria, Paenibacillus polymyxa and Paenibacillus lentimorbus sup-press disease complex caused by root-knot nematode and fusarium wiltfungus. J Appl Microbiol 107:524 –532. http://dx.doi.org/10.1111/j.1365-2672.2009.04238.x.

11. Raaijmakers JM, Mazzola M. 2012. Diversity and natural functions ofantibiotics produced by beneficial and plant pathogenic bacteria. AnnuRev Phytopathol 50:403– 424. http://dx.doi.org/10.1146/annurev-phyto-081211-172908.

12. Guenzi E, Galli G, Grgurina I, Gross DC, Grandi G. 1998. Character-ization of the syringomycin synthetase gene cluster—a link between pro-karyotic and eukaryotic peptide synthetases. J Biol Chem 273:32857–32863. http://dx.doi.org/10.1074/jbc.273.49.32857.

13. Vaillancourt FH, Yin J, Walsh CT. 2005. SyrB2 in syringomycin E bio-synthesis is a nonheme FeII alpha-ketoglutarate- and O2-dependent ha-logenase. Proc Natl Acad Sci U S A 102:10111–10116. http://dx.doi.org/10.1073/pnas.0504412102.

14. Cho H, Kang H. 2012. The PseEF efflux system is a virulence factor ofPseudomonas syringae pv. syringae. J Microbiol 50:79 –90. http://dx.doi.org/10.1007/s12275-012-1353-9.

15. Gross H, Loper JE. 2009. Genomics of secondary metabolite productionby Pseudomonas spp. Nat Prod Rep 26:1408 –1446. http://dx.doi.org/10.1039/b817075b.

16. Gerard J, Lloyd R, Barsby T, Haden P, Kelly MT, Andersen RJ. 1997.Massetolides A H, antimycobacterial cyclic depsipeptides produced bytwo pseudomonads isolated from marine habitats. J Nat Prod 60:223–229.http://dx.doi.org/10.1021/np9606456.

17. Saini HS, Barragán-Huerta BE, Lebrón-Paler A, Pemberton JE,Vázquez RR, Burns AM, Marron MT, Seliga CJ, Gunatilaka AA, MaierRM. 2008. Efficient purification of the biosurfactant viscosin from Pseu-domonas libanensis strain M9 –3 and its physicochemical and biologicalproperties. J Nat Prod 71:1011–1015. http://dx.doi.org/10.1021/np800069u.

18. De Bruijn I, de Kock MJ, de Waard P, van Beek TA, Raaijmakers JM.2008. Massetolide A biosynthesis in Pseudomonas fluorescens. J Bacteriol190:2777–2789. http://dx.doi.org/10.1128/JB.01563-07.

19. Watrous J, Roach P, Alexandrov T, Heath BS, Yang JY, Kersten RD,van der Voort M, Pogliano K, Gross H, Raaijmakers JM, Moore BS,Laskin J, Bandeira N, Dorrestein PC. 2012. Mass spectral molecularnetworking of living microbial colonies. Proc Natl Acad Sci U S A 109:E1743–E1752. http://dx.doi.org/10.1073/pnas.1203689109.

20. Barkei JJ, Kevany BM, Felnagle EA, Thomas MG. 2009. Investigationsinto viomycin biosynthesis by using heterologous production in Strepto-myces lividans. Chembiochem 10:366 –376. http://dx.doi.org/10.1002/cbic.200800646.

21. Quigley NB, Mo YY, Gross DC. 1993. SyrD is required for syringomycinproduction by Pseudomonas syringae pathovar syringae and is related to afamily of ATP-binding secretion proteins. Mol Microbiol 9:787– 801.http://dx.doi.org/10.1111/j.1365-2958.1993.tb01738.x.

22. Hentzer M, Riedel K, Rasmussen TB, Heydorn A, Andersen JB, ParsekMR, Rice SA, Eberl L, Molin S, Høiby N, Kjelleberg S, Givskov M. 2002.Inhibition of quorum sensing in Pseudomonas aeruginosa biofilm bacteriaby a halogenated furanone compound. Microbiology 148:87–102.

23. Koch B, Nielsen TH, Sørensen D, Andersen JB, Christophersen C,Molin S, Givskov M, Sørensen J, Nybroe O. 2002. Lipopeptide pro-duction in Pseudomonas sp. strain dss73 is regulated by components ofsugar beet seed exudate via the Gac two-component regulatory system.Appl Environ Microbiol 68:4509 – 4516. http://dx.doi.org/10.1128/AEM.68.9.4509-4516.2002.

24. De Bruijn I, Raaijmakers JM. 2009. Regulation of cyclic lipopeptidebiosynthesis in Pseudomonas fluorescens by the ClpP protease. J Bacteriol191:1910 –1923. http://dx.doi.org/10.1128/JB.01558-08.

25. Koumoutsi A, Chen XH, Henne A, Liesegang H, Hitzeroth G, Franke P,Vater J, Borriss R. 2004. Structural and functional characterization ofgene clusters directing nonribosomal synthesis of bioactive cyclic lipopep-tides in Bacillus amyloliquefaciens strain FZB42. J Bacteriol 186:1084 –1096. http://dx.doi.org/10.1128/JB.186.4.1084-1096.2004.

26. Dennis JJ, Zylstra GJ. 1998. Plasposons: modular self-cloning minitrans-poson derivatives for rapid genetic analysis of gram-negative bacterialgenomes. Appl Environ Microbiol 64:2710 –2715.

27. Trieu-Cuot P, Derlot E, Courvalin P. 1993. Enhanced conjugative trans-fer of plasmid DNA from Escherichia coli to Staphylococcus aureus andListeria monocytogenes. FEMS Microbiol Lett 109:19 –24. http://dx.doi.org/10.1111/j.1574-6968.1993.tb06137.x.

28. Schweizer HP, Hoang TT. 1995. An improved system for gene replace-ment and xylE fusion analysis in Pseudomonas aeruginosa. Gene 158:15–22. http://dx.doi.org/10.1016/0378-1119(95)00055-B.

29. Koch B, Jensen LE, Nybroe O. 2001. A panel of Tn7-based vectors forinsertion of the gfp marker gene or for delivery of cloned DNA into gram-negative bacteria at a neutral chromosomal site. J Microbiol Methods45:187–195. http://dx.doi.org/10.1016/S0167-7012(01)00246-9.

30. Choi KH, Kumar A, Schweizer HP. 2006. A 10-min method for prepa-ration of highly electrocompetent Pseudomonas aeruginosa cells: applica-tion for DNA fragment transfer between chromosomes and plasmidtransformation. J Microbiol Methods 64:391–397. http://dx.doi.org/10.1016/j.mimet.2005.06.001.

31. Whipps JM. 1987. Effect of media on growth and interactions between arange of soil-borne glasshouse pathogens and antagonistic fungi. New Phytol107:127–142. http://dx.doi.org/10.1111/j.1469-8137.1987.tb04887.x.

32. Gonzalez DJ, Haste NM, Hollands A, Fleming TC, Hamby M, PoglianoK, Nizet V, Dorrestein PC. 2011. Microbial competition between Bacillussubtilis and Staphylococcus aureus monitored by imaging mass spectrom-etry. Microbiology 157:2485–2492. http://dx.doi.org/10.1099/mic.0.048736-0.

33. R Development Core Team. 2009. R: a language and environment forstatistical computing. R Foundation for Statistical Computing, Vienna,Austria.

34. Sambrook J, Russel DW. 2001. Molecular cloning: a laboratory manual,3rd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

Antifungal Peptides from Pseudomonas

March/April 2015 Volume 6 Issue 2 e00079-15 ® mbio.asm.org 9

on August 22, 2020 by guest

http://mbio.asm

.org/D

ownloaded from